During the past year, this laboratory continued studies on the mechanism of HIV entry, as well as development of therapeutic and protective strategies based on the molecules involved in entry. 1) Molecular mechanism of HIV Env-mediated fusion. We examined subunit interactions with the oligomeric structure of HIV Env. Our results indicated that Envs containing different functional defects in gp120 or gp41 can complement one another within the HIV oligomer. They support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. Future work will focus on the implications of these findings for Env function and virus neutralization. 2) Anti-HIV agents based on HIV Env/receptor interactions. a) We continued our work on a novel agent which designed to potently neutralize genetically diverse HIV-1 strains. The agent is a recombinant chimeric protein containing soluble CD4 attached via a long flexible polypeptide linker to a single chain antibody that binds to a CD4-induced eptiope of gp120 involved in binding to coreceptor. We produced small amounts of the protein using vaccinia expression technology. Preliminary results indicate that it potently neutralized infection by a primary HIV-1 isolate. Future studies will focus on strategies to produce larger amounts of the protein sufficient for extensive in vitro analysis, and assessment of protective efficacy in animal models. b) We continue to explore the use of Env-targeted toxins to help eliminate HIV infection when used in combination with highly active antiretroviral therapy (HAART). In vitro studies indicate that a recently designed immunotoxin (3b3-PE38) is significantly more potent in vitro than a previous agent (CD4-PE40). In vitro, 3b3-PE38 efficiently kills HIV-infected cells, and inhibits spreading HIV infection; it displays broad activity against diverse HIV-1 isolates. In vivo studies with rhesus macaques indicate minimal hepatoxicity compared to CD4-PE40, in uninfected and SHIV-infected animals. Future work is focused on developing 3b3-PE38 for Phase I clinical trials in individuals whose virus loads have been effectively suppressed by HAART. 3) Vacccine strategy based on Env/CD4/coreceptor interaction. We extended our analyses of CD4-induced exposure of cryptic neutralization epitopes that are highly conserved in genetically diverse HIV-1 stains. Antibodies against such epitopes potently neutralize Env function if the epitopes are first exposed by CD4 binding. We are exploring whether certain other antibodies might mimic CD4 in exposing these cryptic epitopes. Positive results would suggest vaccine strategies based on elicitation of combinations of antibodies against the inducing and cryptic epitopes.